1. Field of the Invention
In fluorochromosia lymphocytotoxicity, cells labelled with the vital dye carboxyfluorescein diacetate, which imparts a green fluorescence to living cells, but no fluorescence to dead cells, are reacted against cytotoxic monoclonal antibodies in tissue typing trays. The reaction can be assessed visually by an actual viability count of cells counterstained with ethidium bromide, which causes the dead cells to fluoresce with a red fluorescence. This manual method, while being very accurate, is also extremely time consuming.
Reactivity can also be determined by an automated system composed of a microscope mounted photomultiplier tube controlled by a computer, which detects residual green intracellular fluorescence in each well, as a quantative measure of cell viability. In preparing the trays for such automated readings, extracellular fluorescence from lysed cells must be removed by lengthy and complex steps of washing cells in the trays.
It is therefore desirable to find improved ways to simplify the technique which allows for automation, while maintaining the accuracy and efficiency of the automated system.
2. Description of the Prior Art
Bodmer et. al.: Application Of A Fluorochromatic Cyctotoxicity Assay To Human Leukocyte Typing. Histocompatibility Testing, 1967, page 231 describes use of carboxyfluorescein diacetate in tissue typing. Edidin, J. Immunol. (1970) 104:1303 describes counterstaining with ethidium bromide to determine dead cells in a manual method for counting. Bruning et. al., Tissue Antigens (1972) 2:473 describes an automated technique for determining cellular fluorescence.